A two-fold strategy was adopted to identify factors which functionally interact with Prp2p: 1) the yeast two-hybrid system was utilised to identify Prp2p interacting proteins and, 2) the technique of high copy-number suppression screening was used to search for suppressors of dominant negative <i>PRP2</i> mutations. An exhaustive two-hybrid screen with Prp2p as the bait isolated Spp2p, another step 1 splicing factor that was originally identified as a high copy-number suppressor of a <i>prp2-1</i> mutation. Subsequently the reciprocal interaction was observed in a two-hybrid screen with Spp2p as the bait. Surprisingly the most statistically significant result of the Prp2p screen was Sec59p, an endoplasmic reticulum membrane protein involved in core glycosylation. The functional significance of this interaction was investigated by a variety of biochemical and genetic techniques but no evidence implicating Sec59p in pre-mRNA splicing was obtained. An interesting finding from the Spp2p screen was a novel protein Yor093p, which had also been isolated from a two-hybrid screen with Prp17p (a step 2 splicing factor) as the bait. Deletion of the entire open reading frame encoding Yor093p revealed that <i>YOR093c</i> is dispensable for cell growth under all conditions tested. A yeast strain was constructed in which an HA-tagged Yor093p fusion protein was expressed from a chromosomal locus. This strain was subsequently used in coimmunoprecipitation experiments to demonstrate that Yor093p does not stably associate with spliceosomes. A two-hybrid screen with Yor093p as the bait failed to identify any interactions with known splicing factors. The potential role of Yor093p as the bait failed to identify any interactions with known splicing factors. The potential role of Yor093p in pre-mRNA splicing therefore remains unclear. High copy-number suppression screens were performed to isolate putative suppressors of dominant negative <i>PRP2 </i>alleles.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:662139 |
| Date | January 2000 |
| Creators | Smith, Paul Andrew |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12988 |
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